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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4504-4508
Circulating Primitive Stem Cells in Paroxysmal Nocturnal
Hemoglobinuria (PNH) Are Predominantly Normal in Phenotype But
Granulocyte Colony-Stimulating Factor Treatment Mobilizes Mainly PNH
Stem Cells
By
Roderick J. Johnson,
Andy C. Rawstron,
Steve Richards,
Gareth J. Morgan,
Derek R. Norfolk, and
Sheila O'Connor, and Peter Hillmen
From the Haematological Malignancy Diagnostic Service, The General
Infirmary at Leeds, Leeds, UK.
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ABSTRACT |
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic
anemia resulting from a somatic mutation in a hemopoietic stem cell. In
most cases of hemolytic PNH, the majority of the marrow cells are
derived from the PNH clone. Recent evidence has indicated, however,
that the majority of the most primitive peripheral blood stem cells
(PBSCs) in PNH appear to be of normal phenotype. This has led to
tentative suggestions that normal PBSCs could be collected and used for
autologous transplantation. We have investigated this possibility in
four PNH patients by treating them with granulocyte colony-stimulating
factor (G-CSF) in an attempt to mobilize normal progenitors. The
expression of glycosylphosphatidylinositol (GPI)-linked proteins was
analyzed by flow cytometry on mature neutrophils, late stem cells
(CD34+/CD38+), and primitive stem cells
(CD34+/CD38 ). The phenotyping and stem
cell quantitation was performed in steady-state blood and post-G-CSF
administration. The most primitive PBSCs
(CD34+/CD38) were almost all normal before
G-CSF treatment, even when the patients' neutrophils were mainly PNH.
However, after G-CSF, the cells that were mobilized into the peripheral
blood were of a similar phenotype to the mature neutrophils, ie, mainly
PNH. It is possible that PNH-stem cells are preferentially destroyed by complement in the peripheral blood leaving only normal cells in the
circulation. After G-CSF, the PNH cells in the marrow are released into
the blood. Our findings suggest that it would be difficult to collect
sufficient numbers of normal stem cells for autologous transplantation.
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INTRODUCTION |
PAROXYSMAL NOCTURNAL hemoglobinuria (PNH)
is an acquired, clonal hemopoietic disorder that develops when a
somatic mutation of the PIG-A gene occurs in a hemopoietic stem cell in
a susceptible individual.1,2 The PIG-A gene product is
involved in the first identifiable step in the biosynthesis of
glycosylphosphatidylinositol (GPI) anchors by which a
range of molecules are attached to the cell membrane.3
Thus, PNH cells lack GPI-linked molecules including CD59/membrane
inhibitor of reactive lysis (MIRL) and CD55/Decay Accelerating Factor
(DAF), which limit the activity of complement. PNH cells are therefore
abnormally sensitive to complement, leading to hemolysis of red cells
and possibly activation of platelets.4-6 Clinically, this
results in hemolytic anemia and a tendency for thrombosis.
PNH arises in conditions of marrow hypoplasia and there is evidence for
a relative growth advantage of the PNH clone over normal hemopoiesis in
such states.7-10 The mechanism of this selection and the
level of cell maturity at which it operates is unknown. We have
investigated this by using flow cytometry to analyze the proportion of
cells that exhibit a PNH or normal phenotype at different stages of
maturation. Lack of CD59 expression was taken to define the PNH cells.
The combination of CD34 positivity with the level of expression of CD38
was used to identify stem cells of greater or lesser degrees of
maturity. The CD34+/CD38 cell fraction
is known to be enriched for progenitors that can differentiate into
myeloid or lymphoid lineages and represents one of the earliest cell
types identifiable by flow cytometry.11 We have studied the
steady-state peripheral blood stem cells (PBSCs) from four PNH patients
in this way. It has been previously reported in PNH patients that the
most primitive stem cells circulating in the blood are largely of
normal phenotype, even when the marrow CD34+ cells are
almost all PNH.12,13 This leads to the possibility that
these normal cells might be collected from the peripheral blood with a
view to possible reinfusion as an autologous transplant. The baseline
levels of these CD34+ cells are low and therefore an
attempt to mobilize normal PBSCs into the peripheral blood by treatment
with granulocyte colony-stimulating factor (G-CSF) was made. A regimen
similar to that which is used for collection of PBSCs from volunteer
donors in allogeneic peripheral stem cell transplantation was used. The
change in cell numbers and phenotype after administration of G-CSF was
assessed on mature neutrophils and early- and late-stem cells (defined
as CD34+/CD38 and
CD34+/CD38+, respectively).
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MATERIALS AND METHODS |
Patients.
Four patients were investigated. They are identified throughout this
report by a unique patient number that corresponds to their reference
number in the PNH registry maintained in our institution (Table 1). They had all been previously
diagnosed in our department and had classical hemolytic PNH. Diagnostic
criteria included a positive Ham acid lysis test and the demonstration
on red cells and neutrophils of a population of CD59/CD55-deficient
cells by flow cytometry. Baseline clinical status and typical
hematological values for the patients are shown in Table 1. Patient no.
PNH043 was on treatment with Cyclosporin A for aplastic anemia (his
original presentation) and all patients were prophylactically
warfarinized. The subjects all had large PNH clones as judged by
neutrophil surface phenotype (median 87% PNH, range 50 to 97; see
Table 2). Before the enrollment into the
study, full ethical approval was obtained from the hospital ethical
committee according to local practice. Written, informed consent was
then obtained from all patients.
Mobilization regimen.
PBSCs were mobilized using G-CSF alone (Lenograstim; Chugai Pharma,
London, UK) at a dose of 10 µg/kg/d by subcutaneous
injection for 4 consecutive days. This was administered on an
outpatient basis with daily monitoring of hematological parameters.
Phenotypic analysis of neutrophils and CD34 subsets was performed on
steady-state peripheral blood and on day 5 (1 day post-G-CSF).
Phosphatidyl inositol phospholipase-C (PI-PLC) treatment of cells.
PI-PLC (Oxford Glycosystems, Abingdon, UK) was used at a
concentration of 1 U/mL. After washing, 100 µL of PI-PLC was added to
1 × 106 cells in a microtiter well and incubated for
60 minutes before being washed and stained with the appropriate
monoclonal antibodies before fluorescence-activated cell sorting (FACS)
analysis. Appropriate non-GPI-linked controls were tested in parallel
(eg, CD15 for neutrophils).
Flow cytometric analysis.
Leucocytes were prepared by whole blood lysis using a 10-fold excess of
ammonium chloride solution (8.6 g/L) at 37°C for 5 minutes, after
which they were washed and resuspended in FACSFlow (Becton Dickinson,
Oxford, UK). In some cases, CD34+ cell subsets were
analyzed in the mononuclear cells fraction prepared by density-gradient
centrifugation over Lymphoprep (Nycomed, Birmingham, UK).
For antibody staining, 2 to 3 million cells were incubated in
microtiter plates for 20 minutes at room temperature with 10 µL of
each pretitered antibody conjugate per million cells, then washed twice
in FACSFlow. Cells were analyzed on a Becton Dickinson FACSort with
CELLQuest software. The antibodies used in this study were (hybridoma
name in brackets): CD34 PE (Birma-K3); CD45 fluorescein isothiocyanate
[FITC] (9.4); CD38 Cy5 (OKT10); IgG1 control PE/Cy5 (PAP7)-IgG2a
control PE/Cy5 (W6/32HK); and CD59 FITC. CD59-FITC was supplied by
Cymbus Biosciences UK Ltd (Southampton, UK). All other
conjugates were prepared in house.
For CD34 quantitation, cells were incubated with CD34 PE and CD45 FITC,
washed, and resuspended in 150 µL FACSFlow with Propidium Iodide
(PI). A 50,000 event file was collected. Regions were set around the
viable leucocytes (CD45+PI) and
progenitor cells (CD34+SSClo), the latter being
a wide gate including some contaminating events. A further 200 to
500,000 events that satisfied both these regions were then acquired. A
tight CD34 region was then set around the progenitor cells using their
CD34 versus CD45 characteristics. FSC, SSC, and PI of
these gated cells were assessed to ensure that no apoptotic cells or
debris had been included. CD34+ cells were calculated as a
percentage of viable leucocytes. An equivalent number of leucocytes
stained with IgG1/2a control PE, CD45 FITC, and PI were acquired, and
the percentage of control events were deducted to give a final
CD34+ percentage. The absolute numbers of circulating
progenitors were then calculated from the total nucleated cell count.
For CD34+ cell subset analysis, 50,000 events were acquired
and a wide region was set around the progenitor cells
(CD34+SSClo). A 200 to 500,000 event file was
then collected using this gate. To allow simultaneous analysis of CD38
and CD59 expression by progenitor cells, only CD34 and light scatter
characteristics were available for definition of progenitor cells. To
analyze sufficient CD34+ cells, large numbers of total
leucocytes had to be acquired, resulting in increased contamination of
the CD34+ region. To overcome this problem, regions were
used from the CD34 quantitation assay to improve definition. In each
case, the CD34 versus SSC and FSC versus SSC regions were assessed to
determine whether they alone could identify progenitor cells with
greater than 95% accuracy, as defined by their combined CD34, CD45,
PI, and light scatter characteristics. These regions were then applied to allow analysis of CD38 versus CD59 and controls.
Neutrophil alkaline phosphatase (NAP) staining and scoring.
NAP stock substrate was prepared by dissolving 30 mg of naphthol AS
phosphate in 0.5 mL dimethyl formamide and adding 100 mL 0.2 mol/L tris
buffer at pH 9.1. The NAP stain was prepared by adding 2 mg fast blue
BB to 10 mL of this stock substrate. Blood films from
patients and controls were fixed in buffered cold formol acetate for 30 seconds then rinsed. The slides are overlain with the NAP stain for 20 minutes, rinsed, and counterstained with aqueous neutral red for 1 minute. NAP positivity is indicated by the presence of blue/black
granulation in the neutrophil cytoplasm, which is conventionally scored
from 0 to 4. The NAP score for a sample is the sum of the scores of 100 neutrophils assessed by light microscopy. The normal range in our
laboratory is 40 to 100.
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RESULTS |
Samples of blood suitable for analysis were obtained from all four
patients in the steady state and after the administration of G-CSF. One
patient (patient no. PNH046) developed a chest infection after
treatment (presumably unrelated to the G-CSF) and this precipitated a
hemolytic crisis requiring transfusion. The others had no ill effects
apart from mild bone pain related to the growth factor. The peripheral
white blood cell count (WBC) rose in all patients during G-CSF
treatment from a baseline mean of 3.6 × 109/L to a
peak mean of 15.3 × 109/L. The NAP score also
increased in all patients (normal range 40 to 100). The mean NAP score
on day 1 was 41 (range 0 to 123), whereas the mean on day 4 was 162 (range 60 to 250). Three out of the four patients showed a steady
increase that only began to fall again after discontinuing G-CSF,
whereas one patient's score rose initially but began to fall back
towards baseline before G-CSF finished (see
Fig 1).
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| Fig 1.
Graph showing the pattern of increase in the NAP score on
peripheral blood neutrophils in the four PNH patients during
administration of G-CSF.
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The absolute number of peripheral blood CD34+ cells
increased a median of 10-fold (range 4 to 13) from a baseline mean of
0.5 × 106/L (range 0.1 to 0.8) to a peak mean of 4.2 × 106/L (range 1.3 to 9.0). Neither the platelet
count nor the reticulocyte count altered significantly. The phenotypic
analysis of peripheral blood neutrophils and stem cells is summarized
in Table 2. Control samples of peripheral blood from six healthy
volunteers were processed in an identical way and confirmed
observations that CD59 is expressed uniformly on all identifiable
neutrophils and stem cells including the
CD34+/CD38 subset.12,14 The
median absolute CD34 count in normal peripheral blood was 2.5 × 106/L (range 0.9 to 11.0), which is in broad agreement with
other published data.15 Three out of the four patients
(patients no. PNH043, PNH042, and PNH056) showed considerable
differences in the proportion of PNH cells in different cell
compartments and a dramatic change after G-CSF. In steady-state
peripheral blood, these three patients were found to have almost no
detectable PNH cells in the most primitive
(CD34+/CD38) subset (median 0%; range
0% to 7%), whereas in the mature neutrophils, they had large PNH
clones (median 96%; range 50% to 97%). The more mature
CD34+/CD38+ stem cells appeared to have an
intermediate number of PNH cells (median 47%; range 29% to 50%).
After the administration of G-CSF, the pattern changed dramatically.
There was no significant change in the mature neutrophils that remained
largely PNH but both the early and intermediate stem cells became
phenotypically more PNH (median 87%; range 68% to 90% for the
CD34+/CD38+ cells and median 88%; range 51%
to 91% for the CD34+/CD38 cells). The
changes in the CD38 fraction were statistically
significant (P<.05) but those in the CD38+
fraction were not (P<.1). These changes reverted to
steady-state values within a few days of discontinuing G-CSF. One
patient (patient no. PNH046) had approximately 75% PNH cells in all
subsets examined and this percentage did not change significantly after
G-CSF administration. There was no obvious clinical distinction between
this patient and the others to explain this observation.
It has previously been reported by Ninomiya et al16 that
the GPI-linked molecule CD16 can be induced on PNH neutrophils by G-CSF
administration and that this increased expression was sensitive to
PI-PLC cleavage, implying that it was GPI-linked CD16. We also observed
an increase in CD16 staining after G-CSF but the mean fluorescence
intensities of other GPI-linked antibodies on neutrophils (CD55 and
CD59) also increased as did the mean fluorescence intensity of
non-GPI-linked CD15 and of negative control antibodies (CD3 and
IgG1/2). Furthermore, we did not find this apparent increase in
expression on PNH cells sensitive to PI-PLC cleavage, whereas the
expression on the normal residual cells was abolished by PI-PLC as
expected (see Fig 2). We conclude that the
previously reported induction of CD16 on PNH cells after treatment with
G-CSF is probably an artefactual observation.
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| Fig 2.
FACScan histogram plots showing peripheral blood
neutrophils from patient no. PNH046 stained with anti-CD16-PE
(phycoerythrin). The upper two plots are before G-CSF treatment and
show a large PNH population (CD16 ve) and a small, normal, CD16+ve
population. The CD16 is cleaved off by PI-PLC in the upper right plot
but this enzyme has no effect on the negative peak. In the lower two plots, after G-CSF treatment, the negative peak has shifted to the
right but this apparent increase in CD16 expression is not affected by
PI-PLC (see text).
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| |
DISCUSSION |
The changes in hematological parameters and cell surface phenotype in
four PNH patients before and after G-CSF are reported here. The WBC
rose in all patients to a peak mean of 15.3 × 109/L.
This rise is less than that observed in normal volunteers on G-CSF
whose absolute neutrophil count alone reaches mean peak levels of
around 28 × 109/L on similar regimens.15
These findings presumably reflect the underlying hypoplasia in the PNH
patients. There is wide variation in the absolute numbers of peripheral
blood CD34 cells in normal individuals but all four PNH patients in
this study had baseline values that were 10- to 20-fold less than those
generally reported.15 The mean baseline absolute CD34 count
in the peripheral blood was 0.5 × 106/L for the PNH
patients, compared with 8 × 106/L in a series of
normals. After G-CSF, the mean figures were 4.2 × 106/L for the PNH patients and 55 × 106/L for the normals. In both PNH and normal blood this
represents a similar proportionate increase (7- to 9-fold).
The rise in NAP score in normal neutrophils on exposure to G-CSF is
well documented and represents one of a range of stimulatory or priming
effects of this cytokine.17,18 It was interesting to
observe this phenomenon in our patient group because this enzyme is
known to be GPI-linked and consequently the NAP score in such patients
is classically low. Because two of our patients had nearly greater than
95% PNH neutrophils circulating before and after G-CSF, we can be
certain that the increase in NAP score occurred in both PNH and normal
neutrophils alike because the NAP scoring by light microscopy confirmed
that the majority of cells showed increased granulation. Although we
did not formally separate normal and PNH cells for scoring it would be
a mathematical impossibility for the small normal clones in these
patients to account for the increase. As far as we are aware, increase
in the NAP score in PNH patients has not been previously reported.
We have been able to study the relative proportions of normal and PNH
cells occurring at different stages of cell maturity in steady-state
and G-CSF-mobilized peripheral blood in these patients. All four had
substantial PNH clones as assessed by neutrophil phenotype, which
implies that the marrow precursors are also largely PNH. Previous
studies of PNH marrow have supported this view and shown a similar
proportion of affected CD34+ cells to
neutrophils.19 Despite this it has been previously noted
that the great majority of the most primitive peripheral blood stem
cells are in fact of normal phenotype in sharp contrast to the marrow
and the more mature cell types.12,13 Our observations confirm this phenomenon in three out of our four patients. In addition
we have shown a gradient of expression with the most primitive cells
being normal, the more mature stem cells
(CD34+/CD38+) having an intermediate proportion
of PNH cells, and the mature neutrophils being largely PNH. It is not
clear why normal pluripotent cells should circulate selectively in the
peripheral blood of these patients when they are only a small minority
of the marrow precursors. Normal stem cells may be selectively
released, perhaps due to a GPI-linkage deficiency involving adherence
or homing, or they may have a survival advantage over PNH cells when in
the peripheral blood that is not present in the marrow miroenvironment. This phenomenon is marked in the majority of patients and it is intriguing to consider that it may have some relevance to the pathogenesis of PNH.
It has been suggested that normal stem cells could be collected from
the blood and used as a source of normal progenitors for autologous
transplantation in PNH,12,13 but the absolute numbers of
peripheral CD34+ cells in PNH patients is generally very
small. A logical approach would be to mobilize stem cells from such
patients with growth factors, as is done in the management of other
hematological malignancies. However, the number of stem cells mobilized
by G-CSF is disappointing and the vast majority that are released are
of PNH phenotype unlike those that naturally circulate. We found that
after G-CSF the absolute number of CD34+ cells in the
peripheral blood of our patients was a mean of 4.2 × 106/L. (range 1.3 to 9.0) and in the important
CD38 fraction only a minority (median 12%) remained
of normal phenotype. Contrasting this with recommendations for
harvesting in hematological malignancy in which minimum post-G-CSF
levels of around 10 to 20 × 106 CD34+
cells/L of peripheral blood are preferred, it suggests that it would be
difficult to mobilize sufficient numbers of normal progenitors into the
peripheral blood of PNH patients to support transplantation.
| |
ACKNOWLEDGEMENT |
We would like to thank Cymbus Biosciences for provision of some of the
monoclonal antibodies used in this work.
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FOOTNOTES |
Submitted December 1, 1997;
accepted January 26, 1998.
Address correspondence to Dr Roderick J. Johnson, Flat 1, 8 The Avenue, Roundhay, Leeds, UK, LS8 1EH; email: Rodders123{at}aol.com.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract